CN104120148A - Method for synthesizing alpha-pinene or beta-pinene by adopting biological process - Google Patents
Method for synthesizing alpha-pinene or beta-pinene by adopting biological process Download PDFInfo
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Abstract
The invention provides a biological method for synthesizing alpha-pinene or beta-pinene from acetyl coenzyme A and a reconstitution cell capable of synthesizing the alpha-pinene or beta-pinene, wherein the acetyl coenzyme A is obtained from a simple starting material like glucose. The method for synthesizing the alpha-pinene or beta-pinene by adopting a biological process comprises the following steps: transferring acetyl coenzyme A acyltransferase, 3-hydroxy-3-methyl glutaryl coenzyme A synthase, hydroxymethyl glutaryl coenzyme A reductase, mevalonic acid kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-diphosphonic acid decarboxylase, isopentenylpyrophosphate isomerase, geranyldiphosphate synthetase and pinene synthase into an appropriate host cell by adopting metabolic engineering technology, and culturing the obtained reconstitution cell, so that the target product, namely alpha-pinene or beta-pinene can be obtained.
Description
Technical field
The present invention relates to utilize the method for Biological preparation isoprene derivatives.Particularly, the present invention relates to the synthetic α of a kind of biological process-or the method for beta-pinene and can synthesize α-or the reconstitution cell of beta-pinene, wherein acetyl-CoA used is to obtain from the simple parent material such as glucose.
Background technology
Isoprene derivatives---firpene is a kind of important hardware and software platform compound, due to the dimeric high-energy-density of firpene and the very high (energy density: 0.938g/cm of burning net thermal value
3; Burning net thermal value: 39.5MJ/L), be widely used in the synthetic of high-density propellant.In addition, can also be for the synthesis of pharmacological actives, agricultural and domestic biomass actives, functional materials, spices, Terpineol 350, aromatic alcohol, terpinyl acetate, dihydroterpineol, dihydromyrcenol and High-Density Jet etc.
The main method of current industrial acquisition firpene is to adopt high-efficient spiral-screen column to carry out separation and Extraction from gum turpentine or crude sulphate turps.This method exists high to equipment requirements, operational difficulty, and the shortcoming such as energy consumption is large, and efficiency is low causes the waste of a large amount of natural resourcess simultaneously.
The research of carrying out Biological preparation firpene can effectively solve the raw material bottleneck problem that high-density propellant exists.High-density propellant is one of important member of rocket engine fuel, is the propulsion source of jet plane, missile propulsive plant, in guided missile and spationautics development, plays an important role.By the high-density propellant of specific high-density compou nd synthesis, compared with traditional rectifying high-density propellant that to have density larger, combustion heat value is higher, and the better feature of over-all properties causes cost high but be also subject to raw material sources restriction simultaneously.Reduce high-density propellant cost, develop the synthetic technology of continuable high-density propellant, to meet the huge breach of high-density propellant in Military Application, solve high-density propellant insufficient raw material problem and there is its own strategic significance.
In organism, two kinds of natural pathways metabolisms of main existence are carried out the biosynthesizing of isoprene, i.e. mevalonic acid (MVA) approach and methyl E4P (MEP) approach.MVA approach is mainly present in the enchylema of eukaryote, archeobacteria and higher plant, and MEP approach is present in plastid, bacterium and the algae of plant.The final product of this two classes pathways metabolism is all the precursor substance dimethylallylpyrophosphate (dimethylallyldiphosphate, DMAPP) that forms isoprene, passes through afterwards isoprenoid synthase catalysis DMAPP to isoprene and derivative thereof.
Before this, also do not utilize microorganism fermentation to obtain the relevant report of firpene.The present invention has introduced the novel method of utilizing microorganism fermentation to obtain firpene first.
Summary of the invention
The present invention discloses a kind of renewable resources glucose that utilizes for raw material, by biological catalyst, prepares isoprene derivatives---α-or beta-pinene, set up the isoprene derivatives of Sustainable development---α-or beta-pinene synthesis route.
The present invention mainly by genetic engineering means to the synthetic isoprene derivatives of MVA approach---α-or beta-pinene relative enzyme gene in intestinal bacteria, carry out heterogenous expression.Finally in Bacillus coli cells, successfully set up a kind of isoprene derivatives---α-or the biosynthetic pathway of beta-pinene.
Particularly, in first aspect, the invention provides a kind of can be from the synthetic α of acetyl-CoA-or the reconstitution cell of beta-pinene, described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase, wherein said reconstitution cell is transferred to described gene fragment in suitable host cell and prepares by genetic engineering technique.Wherein available host cell is bacterial cell, for example Bacillus coli cells.In embodiment preferably, described reconstitution cell is recombinant Bacillus coli cells, and it can carry out fermentation culture in as the substratum of carbon source comprising glucose, from the synthetic α of acetyl-CoA-or beta-pinene.
Wherein said acetyl-CoA acyltransferase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1); or 2) derive from other bacterium; preferred enterococcus faecalis (Enterococcusfaecalis); or 3) derive from other organism; there is no obvious homology with acetyl-CoA acyltransferase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
Described 3-Hydroxy-3-methylglutaryl CoA A synthase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcusfaecalis), or 3) derive from other organism, there is no obvious homology with 3-Hydroxy-3-methylglutaryl CoA A synthase gene, coding has the nucleotide sequence of the albumen of same or similar function.
Described 3-hydroxy-3-methylglutaryl coenzyme A reductase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcusfaecalis), or 3) derive from other organism, there is no obvious homology with 3-hydroxy-3-methylglutaryl coenzyme A reductase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
Described Mevalonic kinase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, there is no obvious homology with Mevalonic kinase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
Described mevalonic acid-5-phosphokinase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, with mevalonic acid-5-phosphokinase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described mevalonic acid-5-bisphosphate decarboxylase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, with mevalonic acid-5-bisphosphate decarboxylase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
Described isopentenylpyrophosphate isomerase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, there is no obvious homology with isopentenylpyrophosphate isomerase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
Described geraniol ester diphosphate synthase gene source is in intestinal bacteria (Escherichia coli) 1), or 2) derive from abies grandis (Abiesgrandis), preferred abies grandis or 3) derive from other organism, there is no obvious homology with geraniol ester diphosphate synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
Described firpene synthase gene derives from: 1) torch pine (Pinustaeda), 2) abies grandis (Abiesgrandis), 3) Herba Artemisiae annuae (ArtemiSia annua), or 4) derive from other organism, there is no obvious homology with firpene synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
Wherein host cell used is bacterial cell, includes but not limited to Bacillus coli cells.
In a preferred embodiment, by co expression in intestinal bacteria, derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE of enterococcus faecalis (Enterococcusfaecalis), SEQ ID NO:1), 3-Hydroxy-3-methylglutaryl CoA A synthase gene (mvaS, SEQ ID NO:2); Derive from the Mevalonic kinase gene (ERG12 of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), SEQ ID NO:5), mevalonic acid-5-phosphokinase gene (ERG8, SEQ ID NO:6), mevalonic acid-5-bisphosphate decarboxylase gene (ERG19, SEQ ID NO:7), isopentenylpyrophosphate isomerase gene (IDI1, SEQ ID NO:8); Derive from the geraniol ester diphosphate synthase gene (GPPS2 of abies grandis (Abiesgrandis), SEQ ID NO:3) and the α-pinene synthase gene (Pt30, SEQ ID NO:4) that derives from torch pine (Pinustaeda) build the recombinant Bacillus coli cells can utilize glucose degradation intermediate product acetyl-CoA biosynthesizing α-pinene.
In another preferred embodiment, by co expression in intestinal bacteria, derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE of enterococcus faecalis, SEQ ID NO:1), 3-Hydroxy-3-methylglutaryl CoA A synthase gene (mvaS, SEQ ID NO:2); Derive from the Mevalonic kinase gene (ERG12 of yeast saccharomyces cerevisiae, SEQ ID NO:5), mevalonic acid-5-phosphokinase gene (ERG8, SEQ ID NO:6), mevalonic acid-5-bisphosphate decarboxylase gene (ERG19, SEQ ID NO:7), isopentenylpyrophosphate isomerase gene (IDI1, SEQ ID NO:8); Derive from the geraniol ester diphosphate synthase gene (GPPS2 of abies grandis, SEQ ID NO:3) and the beta-pinene synthase gene (QH6, SEQ ID NO:9) that derives from Herba Artemisiae annuae build the recombinant Bacillus coli cells can utilize glucose degradation intermediate product acetyl-CoA biosynthesizing beta-pinene.
Those skilled in the art should understand that, for expressing better in reconstitution cell, the gene order of above-mentioned various enzymes or the nucleotide sequence by its derivative coding with the albumen of same or analogous function can carry out codon optimized according to the codon preference of host cell used.
Those skilled in the art be also to be understood that the gene order of above-mentioned various enzymes or can be cloned in host cell according to conventional molecule clone technology by the nucleotide sequence that its derivative coding has the albumen of same or analogous function.In addition, these nucleotide fragments can also for example, be operably connected with suitable expression controlling elements (, promotor, enhanser etc.).These all, within those skilled in the art's limit of power, do not need to pay performing creative labour.These nucleotide fragments operability connect can also can be without joint by means of joint, and this can carry out appropriate selection according to actual needs by those skilled in the art.
In second aspect, the invention provides the synthetic α of a kind of biological process-or the method for beta-pinene, described method comprises the steps:
A) building can be from the synthetic α of acetyl-CoA-or the reconstitution cell of beta-pinene (, the reconstitution cell of first aspect present invention), described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase;
B) utilize A) described in reconstitution cell in comprising the substratum of glucose, cultivate, with the induction of suitable inductor, after separation and purification, can obtain α-or beta-pinene.
About can be from the synthetic α of acetyl-CoA-or the structure of the reconstitution cell of beta-pinene can be referring to the description of first aspect present invention.
Those skilled in the art should understand that, select suitable constructing host cell well can be from acetyl-CoA synthetic α-or the reconstitution cell of beta-pinene, those skilled in the art can test and (for example determine suitable culture condition according to technology general knowledge or through limited number of time, the parameters such as the temperature of fermentation culture, stirring velocity, pH value, dissolved oxygen rate, fermentation time), also can select suitable inductor, determine the opportunity that adds inductor, etc.This does not need to pay performing creative labour.
Therefore, the invention provides following:
1, the synthetic α of biological process-or method of beta-pinene, described method comprises the steps:
A) building can be from the synthetic α of acetyl-CoA-or the reconstitution cell of beta-pinene, and described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase;
B) utilize A) described in reconstitution cell in comprising the substratum of glucose, cultivate, with the induction of suitable inductor, after separation and purification, can obtain α-or beta-pinene.
2, according to the method described in the 1st; wherein said acetyl-CoA acyltransferase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1); or 2) derive from other bacterium; preferred enterococcus faecalis (Enterococcusfaecalis); or 3) derive from other organism; there is no obvious homology with acetyl-CoA acyltransferase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
3, according to the method described in the 1st, wherein said 3-Hydroxy-3-methylglutaryl CoA A synthase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcusfaecalis), or 3) derive from other organism, there is no obvious homology with 3-Hydroxy-3-methylglutaryl CoA A synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
4, according to the method described in the 1st, wherein said 3-hydroxy-3-methylglutaryl coenzyme A reductase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcusfaecalis), or 3) derive from other organism, there is no obvious homology with 3-hydroxy-3-methylglutaryl coenzyme A reductase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
5, according to the method described in the 1st, wherein said Mevalonic kinase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, there is no obvious homology with Mevalonic kinase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
6, according to the method described in the 1st, wherein said mevalonic acid-5-phosphokinase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, with mevalonic acid-5-phosphokinase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
7, according to the method described in the 1st, wherein said mevalonic acid-5-bisphosphate decarboxylase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, with mevalonic acid-5-bisphosphate decarboxylase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
8, according to the method described in the 1st, wherein said isopentenylpyrophosphate isomerase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, there is no obvious homology with isopentenylpyrophosphate isomerase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
9, according to the method described in the 1st, wherein said geraniol ester diphosphate synthase gene source is in intestinal bacteria (Escherichia coli) 1), or 2) derive from abies grandis (Abiesgrandis), preferred abies grandis or 3) derive from other organism, there is no obvious homology with geraniol ester diphosphate synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
10, according to the method described in the 1st, wherein said firpene synthase gene derives from: 1) torch pine (Pinustaeda), 2) abies grandis (Abiesgrandis), 3) Herba Artemisiae annuae (ArtemiSia annua), or 4) derive from other organism, there is no obvious homology with firpene synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
11. according to the method described in the 1st; steps A wherein) described reconstitution cell proceeds to suitable constructing host cell by genetic engineering technique by the gene fragment of coding acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase and forms; wherein said host cell is bacterial cell, for example Bacillus coli cells.
12, a kind of can be from the synthetic α of acetyl-CoA-or the reconstitution cell of beta-pinene; described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase, wherein said reconstitution cell is transferred to described gene fragment in suitable host cell and prepares by genetic engineering technique.
13, according to the reconstitution cell described in the 12nd, wherein said host cell is bacterial cell, for example Bacillus coli cells.
Beneficial effect of the present invention:
Compare with traditional preparation technology, utilize the route of the synthetic firpene of microorganism catalysis to there is following advantage: (1) is owing to there being narrow spectrum firpene synthetic enzyme, so the product generating has highly selective, industrially can greatly reduce separation costs; (2) raw material using for the glucose that ligocellulose degradation obtains be renewable resources.(3) whole process is to carry out at normal temperatures and pressures, and energy consumption is low.Therefore, utilize biocatalysis means to prepare firpene and will become the inexorable trend of firpene industrial development from now on.In addition, microorganism has fast growth, fermentation period is short, genetic background is clear, be easy to through engineering approaches operation, can utilize the cheap features such as renewable resources, so microorganism has become the effective means of the chemical of production bio-based in recent years as biological catalyst.
Accompanying drawing explanation
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1 utilizes acetyl-CoA biosynthesizing isoprene derivatives---α-or beta-pinene pathways metabolism schematic diagram.
Fig. 2 is pYJM3 plasmid map.
Fig. 3 is pYJM4 plasmid map.
Fig. 4 is pYJM5 plasmid map.
Fig. 5 is that the GC of tunning firpene structure analyzes collection of illustrative plates.
Fig. 6 shows that engineering bacteria YJM28 produces the time curve of α-pinene.The α-pinene output (▲) of engineering bacteria YJM28 and Growth of Cells (■).When engineering bacteria Growth of Cells 12h starts to carry out inducing culture.
Fig. 7 shows the impact of inducing temperature on engineering bacteria α-pinene.Cell OD as engineering bacteria YJM28
600while reaching 0.6-0.9, in substratum, add the IPTG that final concentration is 1mM, at different temperature, carry out respectively inducing culture 29h:25 ℃ (white), 30 ℃ (light gray), 34 ℃ (grey), 37 ℃ (Dark grey).Above-mentioned testing data is mean value in triplicate.
Fig. 8 shows the impact of IPTG concentration on engineering bacteria product α-pinene.Cell OD as engineering bacteria YJM28
600while reaching 0.6-0.9, at inducing temperature, be 30 ℃, under the condition of different final concentration IPTG, induce 66h:0.1mM (■), 0.25mM (◆), 0.5mM (▲), 1mM (●).Above-mentioned testing data is mean value in triplicate.
Fig. 9 is that the GC of tunning firpene structure analyzes collection of illustrative plates.
Embodiment
Below with reference to specific embodiment, further describe the present invention, but it should be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments.
Embodiment 1
By co expression in intestinal bacteria, derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE of enterococcus faecalis (Enterococcus faecalis), SEQ ID NO:1), 3-Hydroxy-3-methylglutaryl CoA A synthase gene (mvaS, SEQ ID NO:2); Derive from the Mevalonic kinase gene (ERG12 of yeast saccharomyces cerevisiae, SEQ ID NO:5), mevalonic acid-5-phosphokinase gene (ERG8, SEQ ID NO:6), mevalonic acid-5-bisphosphate decarboxylase gene (ERG19, SEQ ID NO:7), isopentenylpyrophosphate isomerase gene (IDI1, SEQ ID NO:8); Derive from the geraniol ester diphosphate synthase gene (GPPS2 of abies grandis (Abiesgrandis), SEQ ID NO:3) and derive from the α-pinene synthase gene (Pt30 of torch pine (Pinustaeda), SEQ ID NO:4), utilize glucose degradation intermediate product acetyl-CoA biosynthesizing isoprene derivatives---α-pinene.
The clone of 1.1 foreign genes and the structure of expression vector
1.1.1 the clone of foreign gene
1.1.1.1 the clone of enterococcus faecalis MVA upstream pathways metabolism gene
The mvaS gene (GenBank No.AAG02439) that comes from enterococcus faecalis (Enterococcusfaecalis), mvaE gene (GenBank No.AAG02438) is obtained by chemical synthesis process by Shanghai JaRa company.Be connected with carrier pGH (purchased from Shanghai Jierui Biology Engineering Co., Ltd) respectively afterwards and obtain pGH/mvaS, pGH/mvaE.
1.1.1.2GPPS2, the clone of Pt30 gene
Respectively to coming from Abiesgrandis GPPS2 gene (GenBank No.AF513112.1), Pinustaeda Pt30 gene (GenBank No.AF543530.1) gene order is carried out rare codon analysis (http://www.genscript.com/cgi-bin/tools/rare_codon_analysis), and its rare codon is optimized for to the codon (http://www.jcat.de/) of E.coli preference.GPP synthase gene (GPPS2) after optimization, the rare synthase gene of pinane (Pt30) are served extra large JaRa company and are carried out chemosynthesis, and are connected into and on pGH carrier, form respectively pGH-GPPS2, pGH-Pt30 carrier.
1.1.2 the structure of expression vector
1.1.2.1pYJM24 vector construction
PGH-GPPS2 carrier and pACYCDuet-1 carrier (Novagen) are carried out to double digestion with NdeI and BglII respectively, carrier and the external source fragment ratio of 1: 5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, and then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM24 (pACY-GPPS2) from positive colony, then identifies by restriction enzyme digestion and order-checking.
1.1.2.2pYJM26 vector construction
By pACY-mvaE-mvaS-ispSPa carrier (pYJM20, referring to Jianming Yang, Plos One, 2012DOI:10.1371/journal.pone.0033509) with NcoI and PstI, carry out double digestion respectively with pACY-GPPS2 (pYJM24) carrier, carrier pACY-GPPS2 and the external source fragment mvaE-mvaS ratio of 1: 5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, and then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM26 (pACY-mvaE-mvaS-GPPS2) from positive colony, then identifies by restriction enzyme digestion and order-checking.
1.1.2.3pYJM27 vector construction
PGH-Pt30 carrier and pYJM26 (pACY-mvaE-mvaS-GPPS2) carrier are carried out to double digestion with BglII and XhoI respectively, carrier pACY-mvaE-mvaS-GPPS2 and the external source fragment Pt30 ratio of 1: 5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM27 (pACY-mvaE-mvaS-GPPS2-Pt30) from positive colony, then identifies by restriction enzyme digestion and order-checking.
1.1.2.4pYJM14 (pTrc-low) vector construction
DNA assembling (Lego DNA assembling) method of setting up according to laboratory builds pTrc-low carrier: the method is that a kind of a plurality of DNA fragmentation unwinds by sex change in vitro, annealing is assembled into the fast method of recombinant plasmid, in connection procedure without any Restriction Enzyme.In the process building, by PCR (simple PCR, overlap extension PCR, long tailed primer PCR) method amplifies a series of continuous fragments (successive substrate fragments, SFs), during these fragments of design amplification, need between these adjacent segment, there is one section of long lap, after these fragments are mixed by equimolar ratio example, sex change, annealing.Because the overlap between fragment and fragment is longer, account for the 1/3-2/3 of whole fragment length, the strand that makes the strand after sex change be easy to form with adjacent segment is hybridized, and finally forms ring-type, product after annealing is transformed after intestinal bacteria, finally can form recombinant plasmid.PTrc-low vector construction process is as follows:
Take respectively pTrchis2B skeleton (from Invitrogen company) and yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) genome is template, amplify the Partial Fragment SFIBhB of plasmid pTrchis2B, SFB12hB, and the fragment ERG12 (SF128h12, SFB12h12) of 4 genes in downstream of yeast saccharomyces cerevisiae MVA approach; ERG8 (SF128h8, SF819h8); ERG19 (SF819h19, SF19IhI); IDI (SF19IhI, SFIBhI), then take these fragments or pTrchis2B skeleton is template, goes out to assemble 6 SFs fragment SF128 of plasmid, SF819, SF19I, SFIB, SFB12, SFB by regular-PCR or overlapping pcr amplification.4 gene ERG12, ERG8, ERG19,3 RBS sequences between IDI are when pcr amplification, during by design primer, introduce, and make 4 genes by expressing under single trc (trp-lac promoter) promotor effect.Table 1 is primer and the template of fragment amplification in the process of pTrc-low plasmid construction, and table 2 gathers for primer sequence used.
The primer of table 1 fragment amplification and template
Table?lPCR?templates?and?primers?for?fragment?construction
Table 2pTrc-low builds primer sequence and gathers
Table?2?Primer?sequences?used?to?construct?pTrc-low
1.1.2.5 the structure of pTrc-low plasmid and evaluation
By 6 SFs fragment SF128 of amplification, SF819, SF19I, SFIB, SFB12, SFB mixes in 1.5mL EP pipe by equimolar ratio example, after sealing, (flooding) boiled and made its sex change in the beaker that distilled water is housed, and is then placed under room temperature, to allow its naturally cooling (annealing).Get appropriate volume and connect product Transformed E .coli DH5 α, then coating is added with the LB solid plate of ammonia benzyl mycin, PCR screening positive clone extracts after recombinant plasmid pYJM14 (pTrc-low) from positive colony, then identifies by restriction enzyme digestion and order-checking.
Embodiment 2
The plasmid building is transformed in competent escherichia coli cell, by shake flask fermentation and two kinds of methods of ferment tank, recombinant bacterium is carried out to fermentation culture, utilize chromatography of gases technology tunning to be carried out to the detection of quantitative and qualitative analysis.
The structure of 2.1E.coli recombinant bacterial strain
By pYJM27 (pACY-mvaE-mvaS-GPPS2-Pt30) and pYJM14 (pTrc-low) recombinant plasmid common thermal shock Transformed E .coli BL21 (DE3) competent cell, coat and be added with paraxin and the antibiotic LB solid plate of ammonia benzyl mycin, by PCR, screen and obtain positive colony, obtain thus the engineering colon bacillus that contains pYJM27 and pYJM14.
The cultivation of 2.2 engineering colon bacillus
Ratio by the engineering colon bacillus after activation in 1: 100 is inoculated in the LB liquid medium that contains paraxin and ammonia benzyl mycin, and 37 ℃, shaking culture under 180rpm condition, works as OD
600nmduring for 0.6-0.8, in bacterium liquid, add inductor IPTG to final concentration 0.5mmolL
-1, then proceed at 30 ℃, under 180rpm condition, continue to cultivate.After engineering strain induction 24h, get head space gas 1ml, utilize GC-MS qualitative detection.
2.3 engineering bacterium fermentation tests
Picking mono-clonal is to 50ml M9 seed culture medium (1L M9salts:20g Glucose, 6gNa
2hPO
4, 3g KH
2pO
4, 1g NH
4cl, 0.5gNaCl, 0.24g MgSO
4, 121 ℃ of high pressure steam sterilization 15min.) in, 37 ℃, 180rpm activates spend the night (18-24h).Seed is seeded to and contains 2L fermention medium (19.6g K by 10% inoculum size
2hPO
4.3H
2o; 4.2g citric acid.H
2o; 0.6g ferric ammonium citrate; The 0.8ml vitriol oil; 40g glucose, (NH
4)
6mo
7o
24.4H
2o0.123mg; ZnSO
4.7H
2o0.097mg; H
3bO
40.823mg; CuSO
4.5H
2o0.083mg; MnCl
24H
2o 0.527mg, 4ml1M MgSO
4, 1900ml distilled water) 5L small-sized fermentation tank in, air flow 1.3VVM, rotating speed 400rpm, 37 ℃ are cultured to OD
600be about at 12 o'clock, 0.5mM IPTG, 37 ℃ of abduction deliverings, adjust pH with ammoniacal liquor, control pH 7.0, every 8h, add IPTG one time.The firpene product obtaining carries out qualitative and quantitative analysis by GC-MS to it.In culturing process, remaining glucose in fermented liquid is detected, and add by variable flow the liquid glucose that concentration is 800g/L, maintain remaining sugar concentration below 0.5g/L.Every 4h gets fermented liquid 5ml, measures cell OD
600, glucose concn; Every 15min gets tail gas 1ml, utilizes gas chromatographic detection product isoprene concentration.Until OD no longer changes, till product no longer produces.
Testing conditions: GC system adopts the auspicious rainbow SP-6890 of Shandong Lunan type gas chromatograph, and chromatographic column is HP-INNOWAX column (μ m * 0.2,25m * 250 μ m), and detector is fid detector; 200 ℃ of vaporizer temperature, 230 ℃ of detector temperatures, flow rate of carrier gas: 1ml/min.
Post heating schedule is: 50 ℃ of insulation 0.5min,
4 ℃/min rises to 70 ℃,
25 ℃/min rises to 250 ℃, insulation 5min.
As seen from the figure: fermentation target product accumulative total content is up to 0.97g/L.
Embodiment 3
Different fermentation conditions, as inducing temperature, rotating speed, inductor concentration, nitrogenous source, concentration of substrate, Medium's PH Value and composition proportion etc., can affect the output of tunning firpene.The present invention has detected different inducing temperatures, the impact on firpene output of inductor concentration and nitrogenous source.
The structure of 3.1E.coli recombinant bacterial strain
By pYJM27 (pACY-mvaE-mvaS-GPPS2-Pt30) and pYJM14 (pTrc-low) recombinant plasmid common thermal shock Transformed E .coli BL21 (DE3) competent cell, coat and be added with paraxin and the antibiotic LB solid plate of ammonia benzyl mycin, by PCR, screen and obtain positive colony, obtain thus the engineering colon bacillus that contains pYJM27 and pYJM14.
The impact of the different inducing temperatures of 3.2 research on firpene output
Picking mono-clonal is cultivated activation and is spent the night in 5ml LB bottle, is inoculated in 100ml contains in the antibiotic liquid fermentation medium of Cm+Amp by 1%, and 37 ℃, shaking culture under 180rpm condition, works as OD
600nmduring for 0.6-0.8, in bacterium liquid, add inductor IPTG to final concentration 0.5mmolL
-1, then proceed under differing temps (25 ℃, 30 ℃, 34 ℃, 37 ℃) and carry out inducing culture.At different time points, get 1ml head space gas and carry out GC mensuration.
3.3 impacts of research different IP TG concentration on firpene output
Picking mono-clonal is cultivated activation and is spent the night in 5ml LB bottle, by 1%, being inoculated in 100ml contains in the antibiotic liquid fermentation medium of Cm+Amp, 37 ℃ of shaking culture 4h, when OD600=0.6-1.0 left and right, add different concns IPTG (0.1mM, 0.25mM, 0.5mM, 1mM) under 30 ℃ of conditions, carry out inducing culture.At different time points, get 1ml head space gas and carry out GC mensuration.
Embodiment 4
For studying the difference of different firpene synthase fermentative production firpene ability in engineering bacteria, the present invention replaces the α-pinene synthase gene (Pt30) that derives from torch pine with the beta-pinene synthase gene (QH6) that derives from Herba Artemisiae annuae with identity function, the content of testing goal product firpene.
By co expression in intestinal bacteria, derive from acetyl-CoA acyltransferase gene/3-hydroxy-3-methylglutaryl coenzyme A reductase gene (mvaE of enterococcus faecalis, SEQ ID NO:1), 3-Hydroxy-3-methylglutaryl CoA A synthase gene (mvaS, SEQ ID NO:2); Derive from the Mevalonic kinase gene (ERG12 of yeast saccharomyces cerevisiae, SEQ ID NO:5), mevalonic acid-5-phosphokinase gene (ERG8, SEQ ID NO:6), mevalonic acid-5-bisphosphate decarboxylase gene (ERG19, SEQ ID NO:7), isopentenylpyrophosphate isomerase gene (IDI1, SEQ ID NO:8); Derive from the geraniol ester diphosphate synthase gene (GPPS2 of abies grandis, SEQ ID NO:3) and derive from the beta-pinene synthase gene (QH6 of Herba Artemisiae annuae, SEQ ID NO:9), utilize glucose degradation intermediate product acetyl-CoA biosynthesizing isoprene derivatives---beta-pinene.
The clone of 4.1 foreign genes and the structure of expression vector
4.1.1 exogenous gene cloning
4.1.1.1QH6 the clone of gene
To coming from beta-pinene synthase gene QH6 (GenBank No.ADR64206.1) two ends of Herba Artemisiae annuae (Artemisia annua), add BglII and Xho I double enzyme site, deliver to Shanghai JaRa company and carry out chemosynthesis, be connected on pGH carrier.
4.1.1.2mvaE, mvaS, GPPS2 gene clone
MvaE, mvaS, the clone of GPPS2 gene is the same.
4.1.2 the structure of expression vector
4.1.2.1pYJM28 (pACY-mvaE-mvaS-GPPS2-QH6) vector construction
PYJM26 (pACY-mvaE-mvaS-GPPS2) carrier building in pGH-QH6 carrier and embodiment 1 is carried out to double digestion with BglII and XhoI respectively, carrier pACY-mvaE-mvaS-GPPS2 and the external source fragment QH6 ratio of 1: 5 in molar ratio, 4 ℃ of connections are spent the night or 16 ℃ of connection 4～6h, connect product Transformed E .coli DH5 α, then coating is added with 34 μ gmL
-1the LB solid plate of paraxin, PCR screening positive clone extracts after recombinant plasmid pYJM28 (pACY-mvaE-mvaS-GPPS2-QH6) from positive colony, then identifies by restriction enzyme digestion and order-checking.
4.1.2.2pYJM14 (ppTrc-low) vector construction
PYJM14 (ppTrc-low) carrier construction method is the same.
The structure of 4.2E.coli recombinant bacterial strain
By pYJM28 (pACY-mvaE-mvaS-GPPS2-QH6) and pYJM14 (pTrc-low) recombinant plasmid common thermal shock Transformed E .coli BL21 (DE3) competent cell, coat and be added with paraxin and the antibiotic LB solid plate of ammonia benzyl mycin, by PCR, screen and obtain positive colony, obtain thus the engineering colon bacillus that contains pYJM28 and pYJM14.
The cultivation of 4.3 engineering colon bacillus
Ratio by the engineering colon bacillus after activation in 1: 100 is inoculated in the LB liquid medium that contains paraxin and ammonia benzyl mycin, and 37 ℃, shaking culture under 180rpm condition, works as OD
600nmduring for 0.6-0.8, in bacterium liquid, add inductor IPTG to final concentration 0.5mmolL
-1, then proceed at 30 ℃, under 180rpm condition, continue to cultivate.After engineering strain induction 24h, get head space gas 1ml, utilize GC-MS qualitative detection.
Testing conditions: GC system adopts the auspicious rainbow SP-6890 of Shandong Lunan type gas chromatograph, and chromatographic column is HP-INNOWAX column (μ m * 0.2,25m * 250 μ m), and detector is fid detector; 200 ℃ of vaporizer temperature, 230 ℃ of detector temperatures, flow rate of carrier gas: 1ml/min.
Post heating schedule is: 50 ℃ of insulation 0.5min,
4 ℃/min rises to 70 ℃,
25 ℃/min rises to 250 ℃, insulation 5min.
By detecting quantitatively: fermentation target product accumulative total content is up to 1.67mg/L.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and described, but will be understood by those skilled in the art that, under the condition not deviating from by the spirit and scope of the present invention as defined in the claims, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
Claims (13)
1. the synthetic α of biological process-or method of beta-pinene, described method comprises the steps:
A) building can be from the synthetic α of acetyl-CoA-or the reconstitution cell of beta-pinene, and described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase;
B) utilize A) described in reconstitution cell in comprising the substratum of glucose, cultivate, with the induction of suitable inductor, after separation and purification, can obtain α-or beta-pinene.
2. method according to claim 1; wherein said acetyl-CoA acyltransferase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1); or 2) derive from other bacterium; preferred enterococcus faecalis (Enterococcusfaecalis); or 3) derive from other organism; there is no obvious homology with acetyl-CoA acyltransferase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
3. method according to claim 1, wherein said 3-Hydroxy-3-methylglutaryl CoA A synthase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcusfaecalis), or 3) derive from other organism, there is no obvious homology with 3-Hydroxy-3-methylglutaryl CoA A synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
4. method according to claim 1, wherein said 3-hydroxy-3-methylglutaryl coenzyme A reductase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), or 2) derive from other bacterium, preferred enterococcus faecalis (Enterococcusfaecalis), or 3) derive from other organism, there is no obvious homology with 3-hydroxy-3-methylglutaryl coenzyme A reductase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
5. method according to claim 1, wherein said Mevalonic kinase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, there is no obvious homology with Mevalonic kinase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
6. method according to claim 1, wherein said mevalonic acid-5-phosphokinase gene source is in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 1), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, with mevalonic acid-5-phosphokinase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
7. method according to claim 1, wherein said mevalonic acid-5-bisphosphate decarboxylase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, with mevalonic acid-5-bisphosphate decarboxylase gene do not have obvious homology, but coding has the nucleotide sequence of the albumen of same or similar function.
8. method according to claim 1, wherein said isopentenylpyrophosphate isomerase gene derives from: 1) yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), preferably saccharomyces cerevisiae, or 2) derive from other bacterium, or 3) derive from other organism, there is no obvious homology with isopentenylpyrophosphate isomerase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
9. method according to claim 1, wherein said geraniol ester diphosphate synthase gene source is in intestinal bacteria (Escherichia coli) 1), or 2) derive from abies grandis (Abiesgrandis), preferred abies grandis or 3) derive from other organism, there is no obvious homology with geraniol ester diphosphate synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
10. method according to claim 1, wherein said firpene synthase gene derives from: 1) torch pine (Pinustaeda), 2) abies grandis (Abiesgrandis), 3) Herba Artemisiae annuae (Artemisia annua), or 4) derive from other organism, there is no obvious homology with firpene synthase gene, but coding has the nucleotide sequence of the albumen of same or similar function.
11. methods according to claim 1; steps A wherein) described reconstitution cell proceeds to suitable constructing host cell by genetic engineering technique by the gene fragment of coding acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase and forms; wherein said host cell is bacterial cell, for example Bacillus coli cells.
12. 1 kinds can be from the synthetic α of acetyl-CoA-or the reconstitution cell of beta-pinene; described reconstitution cell comprises following gene fragment: acetyl-CoA acyltransferase, 3-Hydroxy-3-methylglutaryl CoA A synthase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Mevalonic kinase, mevalonic acid-5-phosphokinase, mevalonic acid-5-bisphosphate decarboxylase, isopentenylpyrophosphate isomerase, geraniol ester diphosphate synthase and firpene synthase, wherein said reconstitution cell is transferred to described gene fragment in suitable host cell and prepares by genetic engineering technique.
13. reconstitution cells according to claim 12, wherein said host cell is bacterial cell, for example Bacillus coli cells.
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| CN105838741A (en) * | 2016-03-11 | 2016-08-10 | 青岛农业大学 | Method for fermentation production of isoprenoid compound via peanut shell degradation sugar |
| CN110656056A (en) * | 2019-10-31 | 2020-01-07 | 江南大学 | Construction method of pinene-producing engineering bacteria with high-concentration pinene tolerance |
| CN110951788A (en) * | 2019-12-09 | 2020-04-03 | 中国林业科学研究院亚热带林业研究所 | Application of masson pine α -pinene synthetase in preparation of terpene compounds and products containing terpene compounds |
| CN112391371A (en) * | 2019-08-14 | 2021-02-23 | 中国科学院青岛生物能源与过程研究所 | Tobacco monoterpene synthetase TPS2b, and coding gene and application thereof |
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| CN105838741A (en) * | 2016-03-11 | 2016-08-10 | 青岛农业大学 | Method for fermentation production of isoprenoid compound via peanut shell degradation sugar |
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| CN110951788A (en) * | 2019-12-09 | 2020-04-03 | 中国林业科学研究院亚热带林业研究所 | Application of masson pine α -pinene synthetase in preparation of terpene compounds and products containing terpene compounds |
| CN110951788B (en) * | 2019-12-09 | 2021-06-25 | 中国林业科学研究院亚热带林业研究所 | Application of masson pine alpha-pinene synthetase in preparation of terpene compounds and products containing terpene compounds |
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